The introduction of foreign genes into the germline of recipient animals was first achieved in the early 1980's (Camper (1987) Biotechniques, 5:638-650; Bradley et al. (1984) Nature, 309:255-256; Palmiter and Brinster (1986) Ann. Rev. of Genet., 20:465-499). These types of procedures relied on the microinjection of cloned DNA into the pronuclei of fertilized eggs with subsequent transfer of the eggs into oviducts of pseudopregnant females for gestation (Gordon and Ruddle (1981) Science, 214, 1244-1246). Approximately one to a hundred copies of the microinjected DNA integrate randomly into the genome, usually at a single site, in a fraction of the animals born. These transgenic founder animals usually carry the foreign gene in both somatic cells and germline cells, allowing passage of the transgene to progeny in a Mendelian manner.
The creation of transgenic animals by this and similar methods has proven invaluable as a tool to introduce new and rare genetic characteristics into existing breeds of mammals (Babinet et al. (1989) Genome, 31:938-949; Connelly et al. (1989) Exp. Cell Res., 183:257-276; Scangos and Bieberich (1987) Advances in Genet., 24:285-322). For example, using appropriate regulatory promoter sequences the expression of genes can be directed to the specific tissues or biofluids such as milk or blood of the transgenic animals. Additionally, genes for the proteins of biomedical importance may be fused downstream of specific promoter to express their products either in milk or blood. These proteins can then be isolated from the fluids by common techniques known to those of skill in the art, on a routine basis. Large transgenic species such as cow, goat and sheep may potentially be a valuable reservoir of great quantities of the transgenic protein because of the amount of milk and blood present in these animals. The transgenic animals act as bioreactors or factories for the production of designed, species-specific peptides or proteins or even cells.
However, the generation of these larger species of transgenic animals present some unique challenges (Eyestone (1994) Reprod. Fertil Dev., 6 (5):647-652). In general, the efficiency of gene integration is very poor in these animals. Frequencies of approximately 0.1% for cow and &lt;1.0% for sheep have been reported. Additionally, the survival of microinjected zygotes is low; only 15% in vivo-derived develop into morulae and blastocysts and, of these, only about 18% yield live animals. Thus, more than 1000 zygotes must be injected to produce a single transgenic animal. In addition, the gestation period for large mammals is generally long. These kinds of conditions require that large colonies of donor and recipient animals be maintained. Therefore, given current technology, this type of approach seems logistically and financially prohibitive to implement.
More recently, embryonic stem (ES) cells have been utilized in biotechnological applications such as targeted gene disruption and creation of transgenic mice (Beribault and Kemler (1989) Mol. Biol. Med., 6(6):481-492; Iannaccone et al. (1994) Dev. Biol., 163:288-292; Camper et al. (1995) Biol. of Reprod., 52:246-257). ES cells are pluripotent cells established from normal embryos, and in particular blastocysts. These cells can be cultured and manipulated in vitro and will resume normal development when implanted into blastocysts from foster mothers. Microinjection, electroporation, and retroviral infection have all been successfully used to introduce foreign genes into these cells (Frohman and Martin (1989), (1989) Cell, 56:145-147; and Gordon (1989) Internat. Rev. of Cytol., 115:171-229). The use of gene knockout animals generated from ES cells has been important for studying the hormonal and developmental control of gene expression, for producing animal models of cancer and other diseases and testing the efficacy of gene therapy, and for looking at cell-cell interactions and cell lineage relationships (Camper et al. (1995) Biol. of Reprod., 52:246-257).
One advantage of the ES based method for generating transgenic animals is that cells positive for the presence of the transferred foreign genetic material may be selected for before generation of the transgenic animal. Thus, cells in which homologous recombination of the gene has occurred may be used to establish the transgenic cell line. This propensity towards homologous recombination has been used in creating "knock-outs" or inactivation of endogenous genes as well as to replace defective genes (Capechi (1989) Trends in Genet., 5:70-76; Thompson et al. (1989) Cell, 56:313-321; and Williams et al. (1990) Bone Marrow Transplant., 5:141-144). Mice generated from ES-inoculated blastocysts are chimeric in somatic and germ cells for the selected, novel trait. Inbreeding of heterozygotes permits generation of homozygotes and phenotypic expression of the trait.
A method has now been developed utilizing embryonic stem cells to efficiently generate transgenic animals that highly express an integrated foreign gene. Using this method, chimeric animals are produced that contain ES cells with different integrations of exogenous DNA. Since this method does not rely on homologous recombination, what is produced are compound transgenic animals. The germline of these animals represents a potential source to transmit each independent integration separately to its progeny. Therefore, using the techniques of in vitro fertilization and artificial insemination, the method of the present invention can be used to efficiently and inexpensively create a large number of high expressing animals within a relatively short period of time.